Project Summary My long-term career goal is to become an independent physician-scientist with a research focus in developing novel cellular immunotherapeutics for pediatric blood cancers, especially T-cell acute lymphoblastic leukemia (T-ALL). My clinical experiences as a pediatric oncologist have driven my research interest to develop better therapies for these patients. This proposal outlines a series of preclinical experiments, which in combination with complementary didactics and interdisciplinary mentorship, will provide a promising platform to advance my training in immunology, cancer animal models, and cellular therapy development. This work will be completed under the exceptional mentorship team of Drs. H. Trent Spencer, Edmund K. Waller and Douglas K. Graham, given their collective expertise in cell and gene therapy, translational immunology and leukemia biology. Survival for patients with relapsed T-ALL remains extremely poor. Although allogeneic hematopoietic stem cell transplant (HSCT) provides a chance of cure, attaining disease remission is necessary prior to HSCT, but is seldom achieved. In this research, we capitalize on the promise of gamma delta (??) T cells which unlike the more predominant alpha beta (??) T cells, do not require antigen presentation and identify their targets in a major histocompatibility complex independent manner. Allogeneic ?? T cells, thus have a minimal risk of causing graft-versus-host disease and can be used to create ?off-the-shelf? cellular therapeutics. This is ideal to target T-ALL given the aggressive nature of relapsed disease. Inherent cytotoxic mechanisms used by ?? T cells include recognition of cellular stress molecules such as NKG2D and DNAM-1 receptor ligands and interactions with death pathway receptors Fas and TRAIL-R1/R2 on target cells. Our promising data shows that these markers are overexpressed in T-ALL, making them susceptible to ?? T-cell mediated killing. Furthermore, stress ligand expression can be upregulated by proteasome inhibition, epigenetic modification, and through use of traditional chemotherapeutic agents. We also predict that malignant T-ALL cells can be directly targeted by ?? T cells using CD5-directed chimeric antigen receptors (CARs). CAR T-cell therapy has been challenging in T-ALL given the lack of a specific target antigen on cancerous T cells. However, we have shown that expression of a CAR directed against the pan surface T-cell marker CD5, results in surface CD5 downregulation, thereby reducing the fratricidal effect among CD5-CAR T cells. Additionally, the limited persistence of ?? T cells negates the possibility of life-threatening immune suppression from T-cell aplasia, which may be seen with a CAR-based approach using ?? T cells. Thus, our overarching hypothesis is that the intrinsic cytotoxic activity of ?? T cells can be enhanced and directed towards cancerous T-ALL cells. In Aim 1, we will utilize cellular stress modulation by three distinct mechanisms to sensitize T-ALL to ?? T cell-mediated cytotoxicity, whereas in Aim 2, we will optimize the functionality of CD5-CAR ?? T cells against this disease.